The invention relates generally to methods and systems for analyzing data from a Polymerase Chain Reaction (PCR) amplification reaction, and more particularly to methods and systems for identifying the quantitation cycle (Cq) for a PCR amplification reaction.
PCR is a powerful technique used to amplify genetic material. Quantitative PCR (q-PCR) is a technique used to quantify the amount of a targeted genetic material initially present in a sample. For example, under certain conditions, a cell may alter its expression of a target gene. Q-PCR allows a researcher to quantify the effect of different conditions on the expression of a target gene.
Q-PCR techniques rely on some method of detecting a change in the quantity of a PCR product over the course of a multitude of PCR cycles. Q-PCR techniques generally utilize fluorescent probes that increase in fluorescence relative to the amount of PCR product produced during each amplification cycle. Detecting fluorescence attributable the PCR product is complicated by the presence of background fluorescence in the PCR reaction chamber. Thus, an important factor affecting the accuracy and reproducibility of q-PCR data is identifying the amplification cycle wherein the fluorescent signal attributable to the amplification of the PCR product is detectable above background fluorescent signal.
To this end, conventional q-PCR analytical techniques first identify a threshold fluorescence value, which is then used to identify the Cq. The threshold value is a minimum fluorescence signal value wherein the fluorescence signal is attributable to the amplification of the PCR product. The Cq is then identified as the PCR cycle where the fluorescence from the amplified PCR product is greater than the threshold value.
A variety of conventionally methods for identifying the threshold value are available. For example, in one method, the average fluorescence of a background region is added to a multiple of the standard deviation for the average fluorescence of the background region of a PCR amplification plot. Other methods use complex algorithms and statistical analyses of the amplification data to identify the threshold. These indirect methods of identifying the Cq based on the threshold can yield variable and inaccurate results that are difficult to reproduce.
However, methods and systems of directly identifying the Cq for data from a PCR amplification reaction that do not rely on identifying a threshold value are needed.